Development of analytical method for determination of 1,4-dioxane in cleansing products.

OBJECTIVE: 1,4-Dioxane is a toxic by-product formed during the synthesis of surfactants used in finished cosmetic products. There are no set permissible levels of toxic impurities in finished cosmetic products in Japan. In this study, we have established a simple and sufficiently precise analytical method to determine the activity of 1,4-dioxane in finished cosmetic cleansing products. METHODS: This method involves the standard addition approach and headspace-gas chromatography/mass spectrometry without pre-conditioning. RESULTS: Fifteen cleansing products that are sold in the Japanese market, such as shampoo, hand soap, and dishwashing liquid, were analyzed, and 1,4-dioxane was detected at a concentration of a few micrograms per gram of the product in almost all of them. The concentration of 1,4-dioxane in two dishwashing liquid products was high. The maximum concentration of 1,4-dioxane in all of the cleansing products was below 10 µg g(-1) , which is a limit that is thought to be safe and technically achievable through the application of good manufacturing practices. Since 1,4-dioxane is formed during the synthesis of polyoxyethylene ether sulfate, it was detected at high concentrations in cleansing products that contained a lot of polyoxyethylene ether sulfate. CONCLUSION: Control of the synthesis of polyoxyethylene ether sulfate can be effective in reducing the concentration of 1,4-dioxane in cleansing products.

In vivo delivery of interferon-α gene enhances tumor immunity and suppresses immunotolerance in reconstituted lymphopenic hosts.

T cells recognize tumor-associated antigens under the condition of lymphopenia-induced homeostatic proliferation (HP); however, HP-driven antitumor responses gradually decay in association with tumor growth. Type I interferon (IFN) has important roles in regulating the innate and adaptive immune system. In this study we examined whether a tumor-specific immune response induced by IFN-α could enhance and sustain HP-induced antitumor immunity. An intratumoral IFN-α gene transfer resulted in marked tumor suppression when administered in the early period of syngeneic hematopoietic stem cell transplantation (synHSCT), and was evident even in distant tumors that were not transduced with the IFN-α vector. The intratumoral delivery of the IFN-α gene promoted the maturation of CD11c(+) cells in the tumors and effectively augmented the antigen-presentation capacity of the cells. An analysis of the cytokine profile showed that the CD11c(+) cells in the treated tumors secreted a large amount of immune-stimulatory cytokines including interleukin (IL)-6. The CD11c(+) cells rescued effector T-cell proliferation from regulatory T-cell-mediated suppression, and IL-6 may have a dominant role in this phenomenon. The intratumoral IFN-α gene transfer creates an environment strongly supporting the enhancement of antitumor immunity in reconstituted lymphopenic recipients through the induction of tumor-specific immunity and suppression of immunotolerance.

Mechanism of the rejection of major histocompatibility complex class I-disparate murine skin grafts: rejection can be mediated by CD4+ cells activated by allo-class I + II antigen in CD8+ cell-depleted hosts.

In the preceding article, we analyzed the immunohistochemical rejection mechanism of major histocompatibility complex (MHC) class I (H-2K)-disparate murine skin grafts, and showed that only CD8+ cells infiltrated at the site of the epithelial tissue of MHC class I-disparate graft. We also showed that perfect survival of MHC class I-disparate grafts were attained in thymectomized recipients treated with anti-Lyt-2 monoclonal antibody. In this report, we showed that these long-surviving allo-class I grafts were rejected in the absence of CD8+ cells by stimulation with allo-MHC class I + II-disparate graft as the second stimulation. Furthermore, it was immunohistochemically revealed that under that condition, a large number of CD4+ cells infiltrated into the epithelial tissue of these long-surviving class I grafts, which were going to be rejected 2-5 d after the transplantation of a second graft with MHC class I + II difference. This result directly shows that CD4+ cells are able to became effectors for the rejection of allo-MHC class I (H-2K) skin graft.

Dendritic cell maturation overrules H-2D-mediated natural killer T (NKT) cell inhibition: critical role for B7 in CD1d-dependent NKT cell interferon gamma production.

Given the broad expression of H-2 class Ib molecules on hematopoietic cells, antigen presentation pathways among CD1d expressing cells might tightly regulate CD1d-restricted natural killer T (NKT) cells. Bone marrow-derived dendritic cells (BM-DCs) and not adherent splenocytes become capable of triggering NK1.1(+)/T cell receptor (TCR)(int) hepatic NKT cell activation when (a) immature BM-DCs lack H-2D(b)-/- molecules or (b) BM-DCs undergo a stress signal of activation. In such conditions, BM-DCs promote T helper type 1 predominant CD1d-restricted NKT cell stimulation. H-2 class Ia-mediated inhibition involves more the direct H-2D(b) presentation than the indirect Qa-1(b) pathway. Such inhibition can be overruled by B7/CD28 interactions and marginally by CD40/CD40L or interleukin 12. These data point to a unique regulatory role of DCs in NKT cell innate immune responses and suggest that H-2 class Ia and Ib pathways differentially control NKT cell recognition of DC antigens.

Reconstruction of three-dimensional human skin model composed of dendritic cells, keratinocytes and fibroblasts utilizing a handy scaffold of collagen vitrigel membrane.

We previously we attempted to make a three-dimensional human skin model consisting of three different cells, dendritic cells, keratinocytes and fibroblasts (KDF-Skin) to evaluate immunoreactions in human skin; however, this model had various problems; for example (1) the incubation period for the construction of this model is long (about three weeks); (2) to construct the collagen gel, high amounts of fibroblasts are needed; and (3) the horny layer of keratinocytes in this skin model is thinner than that of keratinocytes in real human skin. In order to overcome these problems, a new three-dimensional human skin model utilizing a handy scaffold of collagen vitrigel membrane (VG-KDF-Skin) was constructed. As a result, after 14 days incubation, the epidermis layer of normal human keratinocytes was thicker than the keratinocyte layer of KDF-Skin. The incubation period for VG-KDF-Skin construction was 7 days shorter than that of KDF-Skin, and the number of fibroblasts needed to seed VG-KDF-Skin was four times fewer than that of KDF-Skin. After the application of sensitizers such as DNCB, VG-KDF-Skin induced the expression of CD86 and cytokine release. These results suggest that the new three-dimensional human skin model consisting of dendritic cells, keratinocytes, fibroblasts and collagen vitrigel membrane was more useful for alternative animal testing than the KDF-Skin model.

Insufficient ex vivo expansion of Valpha24(+) natural killer T cells in malignant lymphoma patients related to the suppressed expression of CD1d molecules on CD14(+) cells.

BACKGROUND: Valpha24(+) natural killer T (NKT) cell is a human counterpart of mice Valpha14(+) NKT cell that has a regulatory role for innate and acquired potential antitumor activity. The efficient expansion of NKT cells is an obstacle to the clinical application of Valpha24(+) NKT cells for immunotherapy. METHODS: We used mononuclear cells (MNC) obtained from the peripheral blood (PB) of normal healthy donor (HD) and malignant lymphoma (ML) patients before and after granulocyte colony-stimulating factor (G-CSF) treatment. MNC were cultured for 12 days with alpha-galactosylceramide (100 ng/mL) and interleukin-2 (IL-2; 100 U/mL). RESULTS: The fold expansion of Valpha24(+) NKT cells was higher in HD than in ML patients (208 versus 0.00), despite comparable numbers of Valpha24(+) NKT cells before culture. G-CSF administration enhanced the predominance of Valpha24(+) NKT cell fold expansion in HD compared with ML patients (1935 versus 1.95). After treatment with G-CSF, the expression of CD1d molecules was up-regulated in CD14(+) cells from HD but not ML patients. The fold expansion of Valpha24(+) NKT cells and CD1d expression on CD14(+) cells was strongly correlated in both HD and ML patients (r(2)=0.84). However, replacement of a patient's CD14(+) cells with HD cells did not increase the efficacy of Valpha24(+) NKT cell expansion. DISCUSSION: G-CSF-mobilized PB from ML patients has inhibitory characteristics for Valpha24(+) NKT cell expansion as a result of both monocytes and Valpha24(+) NKT cells. Multiple procedures would be needed for the expansion of patients' Valpha24(+) NKT cells.

Details of an isolation method for hepatic lymphocytes in mice.

The liver comprises a unique lymphocyte population, i.e., extrathymic alpha beta T cells with TcR of intermediate intensity. In the present study, we attempted to determine what pretreatments were appropriate to isolate hepatic mononuclear cells (MNC) containing such intermediate alpha beta TcR cells in mice. Hepatic MNC were isolated from untreated mice and mice subjected to either bleeding or liver perfusion, and the intermediate alpha beta TcR cells in each preparation were identified. For reasons of simplicity, cell purity and cell yields, hepatic lymphocytes should be obtained from mice subjected to total bleeding. Additional information on extrathymic alpha beta T cells obtained by using the recommended method is also presented.

Rejection of cultured keratinocyte allografts in presensitized mice.

The fate of cultured keratinocyte (KC) allografts remains controversial. Although prolonged survival of cultured KC allografts in naive mice has been reported, the detailed mechanisms remain undetermined. Furthermore, it was also reported that in the human cultured KC allografts do not survive permanently, and they are rapidly replaced by recipient cells. In the present study, we have addressed this issue and obtained findings that cultured KC allografts survive for a prolonged period in naive mice under the conditions in which reepithelization by recipient cells is prevented. However, the same cultured KC allografts were rejected when they were grafted onto recipients primed with allogeneic spleen cells or full-thickness skin grafts. To clarify the mechanisms behind these findings, the allostimulatory ability of cultured KC and their susceptibility to alloreactive cytotoxic T cells were examined in vitro. In a mixed epidermal cell-lymphocyte reaction, cultured KC were unable to induce allospecific proliferative responses of naive T cells. On the other hand, primed T cells from presensitized mice showed weak but significant proliferative responses against allogeneic KC. It also was confirmed that cultured KC are susceptible to lysis by alloreactive cytotoxic T cells. These data indicate that prolonged survival of cultured KC allografts in naive mice is attributable to a defect in the afferent, but not the efferent, phase of the rejection process that is caused by the weak allostimulatory ability of cultured KC. This assumption was also supported by the finding that spleen cells from the recipient mice bearing long-surviving KC allografts retain in vitro responsiveness against stimulator cells syngeneic to the grafted KC. Taken together, these findings indicate that long-term survival of cultured KC allografts in naive mice may be due solely to the weak allostimulatory ability of cultured KC, but not to loss of susceptibility to alloreactive cytotoxic T cells after culture of KC or induction of tolerance in the recipient mice bearing KC allografts.

Mouse NK1.1+ cytotoxic T cells can be generated by IL-2 exposure from lymphocytes which express an intermediate level of T cell receptor.

NK-like T cells which express the NK1.1 molecule and CD3 (or TCR) of intermediate level (CD3int or TCRint cells) were recently demonstrated to be present in various immune organs, and to have NK-like cytotoxic activity against NK target cells. In this study, we investigated whether NK1.1- T cells could express NK1.1. We found that NK1.1+ TCRint cells were much more abundant in the liver (20%) than in the spleen (2%). When hepatic and splenic mononuclear cells (MNCs) were cultured either in the absence of IL-2 or in the presence of CD3/TCR cross-linking, the original NK1.1+ TCRint cells disappeared. However, when they were cultured in the presence of a high dose of IL-2 for 4 days, a new type of NK1.1+ T cell was formed to the extent of approximately 15-20%, and the liver and spleen contained similar percentages of this new type of NK1.1+ T cells. The phenotypes of the original and the new type of NK1.1+ T cells were clearly distinct. The freshly obtained NK1.1+ TCRint cells consisted of double-negative (DN) CD4-CD8- cells and single-positive (SP) CD4+ cells, whereas the new type of NK1.1+ T cells predominantly consisted of DN CD4-CD8- cells and SP CD8+ cells and expressed a high level of CD3 (CD3high or TCRhigh cells). When NK1.1- cells or IL-2 receptor beta-chain (IL-2Rbeta)- cells were isolated from the liver and spleen, and cultured in the presence of IL-2 for 4 days, NK1.1+ T cells were generated from NK1.1- cells, but not from IL-2Rbeta- cells. Our results suggested that the NK1.1- cells, but not IL-2Rbeta- cells, contained the precursor of IL-2-stimulated NK1.1+ TCRhigh cells. When purified NK1.1- IL-2Rbeta+ TCRint cells were cultured in the presence of IL-2 for 4 days, approximately 10% of the cells became NK1.1+ TCRhigh cells. Approximately 60% of the purified NK1.1+ TCRint cells lost NK1.1 expression. The IL-2-stimulated NK1.1+ TCRhigh cells that had arisen from NK1.1- TCRint cells exerted an NK cell-like cytotoxic activity similar to that of the original NK1.1+ T cells. Thus, NK1.1- TCRint cells could express NK1.1 and exert NK-like cytotoxic activity regardless of their origin. It appears that NK1.1+ TCRhigh cells can only be induced through an IL-2-stimulation pathway but not via CD3/TCR cross-linking.

Establishment and usefulness of an anti-human CD57 IgG1 monoclonal antibody.

In the present study we established a new monoclonal antibody, JNK-1, which recognizes all cells recognized by CD57/HNK-1 mAb. JNK-1 and CD57 mAbs inhibited the binding of each other, suggesting that the molecules they recognize are either identical or sufficiently close to cause steric hindrance in the binding assay. JNK-1 mAb detected the 110-kDa protein, which is identical to the protein recognized by CD57/HNK-1 mAb in Western immunoblot analysis combined with immunoprecipitation. Therefore, JNK-1 mAb appears to recognize homogeneous molecules identified by the currently available CD57 mAb. Notably, JNK-1 mAb is composed of mouse IgG1 heavy chains, and thus can be used easily in immunoprecipitation, which cannot easily be performed with the available CD57 mAb because it is an IgM isotype. Thus, JNK-1, which is an IgG isotype, may present a useful tool to elucidate the CD57 protein.

The role of nitric oxide in cholinergic neurotransmission in rat trachea.

1. We have investigated the role of nitric oxide (NO) in cholinergic contraction in rat trachea. 2. Methylene blue (10 nM to 30 microM) potentiated cholinergic contraction induced by electrical field stimulation (EFS) at 5 Hz in a concentration-dependent fashion. At a concentration of 30 microM, methylene blue decreased responses to log EFS frequency, producing 50% of maximum contraction from a control value of 0.74 +/- 0.09 Hz to 0.30 +/- 0.05 Hz without a significant effect on concentration-response curves to acetylcholine (ACh). 3. NG-monomethyl-L-arginine (L-NMMA; 100 microM) also potentiated cholinergic contraction induced by EFS at 5 Hz (131.5 +/- 4.6% of control) without having any effect against ACh (3 microM)-induced contractions. Likewise, L-NMMA (100 microM) significantly increased EFS (5 Hz)-evoked release of ACh from tracheal segments into the bath solution (51.4 +/- 4.0 pmol ml-1 in the presence of L-NMMA and 35.0 +/- 1.8 pmol ml-1 in the absence of L-NMMA, respectively). 4. Administration of NO (present in acidified solution of NaNO2) (1 nM to 10 microM) and sodium nitroprusside (100 nM to 10 microM) concentration-dependently reduced EFS (5 Hz)-induced cholinergic contractions without having a significant effect on ACh (3 microM)-induced contractions. These results were unaffected by prior exposure of the tissues to L-NMMA (100 microM). 5. Dibutyryl cyclic GMP (3 mM) also reduced cholinergic contractions induced by EFS at 5 Hz (70.1 +/- 3.6% of control) without any significant effect on ACh (3 microM)-induced contractions. 6. Pretreatment of tissues with capsaicin (30 microM) or a-chymotrypsin (1 u ml-') failed to inhibit methylene blue (30 microM)-induced potentiation of responses to EFS at 5 Hz.7. These results suggest that an endogenous NO-like factor may mediate prejunctional inhibition of cholinergic contraction through a cyclic GMP-dependent mechanism in rat trachea.

Up-regulation of calcitonin gene-related peptide receptors underlying elevation of skin temperature in ovariectomized rats.

We investigated the mechanism for the augmentation of the calcitonin gene-related peptide (CGRP)-induced elevation of skin temperature in ovariectomized (OVX) rats. I.v. injection of alphaCGRP (10 micro g/kg) elevated skin temperature of the hind paws. The elevation was significantly greater in OVX rats than in sham-operated rats and was inhibited by pretreatment with human CGRP(8-37) (100-1000 micro g/kg i.v.), a CGRP receptor antagonist, in a dose-dependent manner. In addition, ovariectomy not only potentiated vasorelaxation due to alphaCGRP but increased the number of CGRP receptors in mesenteric arteries. Further, the plasma concentration of endogenous CGRP was significantly lower in OVX rats. These results suggest that the low concentration of plasma CGRP due to ovarian hormone deficiency may induce the increase in the number of CGRP receptors due to up-regulation. Therefore, the increased number of CGRP receptors may be responsible for potentiation of exogenous alphaCGRP-induced elevation of skin temperature in OVX rats. The mechanism underlying the hot flashes observed in menopausal women may also involve, in part, the up-regulation of CGRP receptors following ovarian hormone deficiency.

Effects of the Japanese herbal medicine Keishi-bukuryo-gan and 17beta-estradiol on calcitonin gene-related peptide-induced elevation of skin temperature in ovariectomized rats.

The effects of a Japanese herbal medicine, Keishi-bukuryo-gan, and 17beta-estradiol on calcitonin gene-related peptide (CGRP)-induced elevation of skin temperature were investigated in ovariectomized (OVX) rats. Ovariectomy not only potentiated CGRP-induced elevation of skin temperature and arterial vasorelaxation but also induced a lower concentration of endogenous CGRP in plasma and up-regulation of arterial CGRP receptors, suggesting that lowered CGRP in plasma due to ovarian hormone deficiency increases the number of CGRP receptors and consequently amplifies the stimulatory effects of CGRP to elevate skin temperature. Oral Keishi-bukuryo-gan (100-1000 mg/kg, once a day for 7 days) restored a series of CGRP-related responses observed in OVX rats by normalizing plasma CGRP levels in a dose-dependent manner as effectively as s.c. injection. 17Beta-estradiol (0.010 mg/kg, once a day for 7 days). However, Keishi-bukuryo-gan did not affect the lower concentration of plasma estradiol and the decreased uterine weight due to ovariectomy, although the hormone replacement of 17beta-estradiol restored them. These results suggest that Keishi-bukuryo-gan, which does not confer estrogen activity on plasma, may be useful for the treatment of hot flashes in patients for whom estrogen replacement therapy is contraindicated, as well as menopausal women.

Recipient-derived T cells participate in autoimmune-like hepatic lesions induced by graft-versus-host reaction.

Autoimmune-like hepatic lesions were induced by injection of CD4+ T cells from B10.Thy-1.1 mice into MHC class II-disparate (B10.Thy-1.1 x bm12)F1 mice. Hepatic lesions characterized by mononuclear cell accumulation in the portal area of the central vein and around interlobular bile ducts were observed in these recipients. The morphologic features of the lesions resembled primary biliary cirrhosis. The origin of T cells invading at the site of the hepatic lesions was immunohistochemically analyzed. It was shown that many recipient-derived T cells were present at the lesions and that some of them infiltrated the bile duct epithelia. Furthermore, the lesions were weakened when recipient-type T cells were depleted by thymectomy and the administration of anti-Thy-1.2 monoclonal antibody. Recipient-derived T cells were observed to take part in the formation of autoimmune hepatic lesions. These findings suggest the possibility that the tolerance of self-reactive T cells is abrogated by the graft-versus-host reaction.

Histological characteristics of lupus nephritis in F1 mice with chronic graft-versus-host reaction across MHC class II difference.

Renal lesions at the chronic phase of MHC class-II-disparate graft-versus-host reaction (GVHR) were examined. To induce GVHR, C57BL/6 (B6) spleen cells were injected twice into either (B6 x bm12)F1 (class-II-disparate), (B6 x bm1)F1 (class-I-disparate) or (bm1 x bm12)F1 mice (class-I + II-disparate). For comparison, (C57BL/10 x DBA/2)F1 (BDF1) mice injected with DBA/2 spleen cells were also used. (B6 x bm12)F1 and BDF1 recipients showed marked elevation of anti-DNA antibodies, circulating immune complexes (CIC) and the number of immunoglobulin producing cells (IgPC). At 20 weeks after cell injection, severe immune complex glomerulonephritis (ICGN) was observed in (B6 x bm12)F1 recipients, but was far less severe in (bm1 x bm12)F1 recipients and was not observed in (B6 x bm1)F1 recipients. ICGN was also observed in BDF1 recipients at 12 weeks after cell injection. By immunofluorescent microscopy, IC deposition was detected along the capillary loops and also in the mesangial area in (B6 x bm12)F1 recipients, while BDF1 recipients showed only a capillary pattern. By light microscopy, the renal lesion of (B6 x bm12)F1 recipients appeared similar to those of BDF1 recipients. Histologically, (B6 x bm12)F1 recipients serve as a good model for lupus glomerulonephritis induced by class-II-disparate GVHR.

Effect of heat treatment of poly(L-lactide) on the response of osteoblast-like MC3T3-E1 cells.

Poly(L-lactide) (PLLA) products are molded by heat extrusion. These treatments may change chemical properties and biological response of the PLLA to cells. In this study, the effect of heat treatment of PLLA on osteoblast proliferation and differentiation was examined in vitro. Osteoblast-like MC3T3-E1 cells were cultured for 2 weeks on the PLLA subjected to various heating temperature and time combinations. The protein, DNA, and hydroxyproline (HYP) contents and alkaline phosphatase (ALP) activity of cells cultured on the untreated (non-heated) PLLA with a weight average molecular weight (Mw) of 1,000,000 (high Mw PLLA) were not significantly different from those of cells cultured on glass. The activation of osteoblast differentiation by the high Mw PLLA was weak. In contrast, increases in ALP activity and HYP content were found for cells cultured on the PLLA heated at a high temperature of 200 or 250 degrees C. Heat treatment of high Mw PLLA increased differentiation of MC3T3-E1 cells cultured upon it. Significant degradation of PLLA (decrease in molecular weight and increase in molecular weight distribution) were observed following heat treatment. The Mw of PLLA decreased from 1,000,000 to below 20,000, and 14.4 microg of L-lactic acid was released from 10 mg of PLLA by heating at 250 degrees C. Therefore, the effect of low Mw chemicals, which were expected to be the degradation products of high Mw PLLA after heat treatment, on MC3T3-E1 cell activities was examined. Increases in the protein, DNA and HYP amounts and ALP activity for cells cultured with L-lactide or L-lactic acid were observed at 100 microg/ml, but not at 10 microg/ml. When the cells were cultured on the low Mw PLLA (Mw 20,000), their biological parameters also increased. Twelve micrograms of L-lactic acid released from 10 mg of the low Mw PLLA during 2 weeks incubation. The concentration of L-lactic acid in the incubation solution of low Mw PLLA or heat-treated PLLA was too small to cause cell activation. These results suggested that increases in osteoblast differentiation on the heat-treated PLLA was not to due to soluble degradation chemicals, such as L-lactic acid, rather than the remaining low Mw PLLA.

Sensitization potential of gold sodium thiosulfate in mice and guinea pigs.

Since gold sodium thiosulfate (GST) has been included in a standard patch test series for diagnosis of allergic contact dermatitis from gold, the incidence of patients showing positive reactions to gold is increasing. However, there were little reports on induction of gold sensitization in animals. In this study, we have examined the sensitization potential of GST using mice and guinea pigs. In the guinea pig maximization test, 2 or 6 out of 10 animals showed positive skin responses, mainly edema, by challenge with 2% or 5% GST in 50% ethanol solution, respectively. In the mouse ear swelling test, positive ear swelling (20% greater increase in ear thickness) after challenge with GST was shown in 2 out of 6 mice those previously treated with GST. Topical exposure of mice to GST in 70% dimethylsulfoxide solution induced small increases in the lymph node weight and the lymph node cell (LNC) number in the murine local lymph node assay (LLNA). A greater degree of LNC responses were observed in the sensitive mouse lymph node assay (SLNA) compared with the LLNA, but the stimulation index of total lymph node response by GST was not so high. From these results, GST was identified as a contact allergen, but the sensitization potential was not so strong. In the mouse IgE test, treatment of mice with GST resulted in a statistically significant increase in the serum IgE antibody concentration that associated with immediate-type hypersensitivity reaction. It may suggest that the sensitization responses from gold would appear not only at the contact site but also systematically.

Cytotoxicity of medical materials sterilized with vapour-phase hydrogen peroxide.

A new sterilization system using vapour-phase hydrogen peroxide (VPHP) was recently developed. The cytotoxicity of various medical materials sterilized by the VPHP sterilization system was investigated. After VPHP sterilization, polystyrene, polyurethane (PU8), blend material of silicone and polyurethane (Sil/PU6), poly(methyl methacrylate) (PMMA), fluorosilicone acrylate and poly(2-hydroxyethyl methacrylate) (HEMA) showed strong cytotoxicity, whereas polyethylene and polypropylene did not. Although the cytotoxic potential of most materials is reduced by extension of the aeration time, HEMA and PMMA still retained strong cytotoxicity after 12 h aeration. Addition of catalase to the cell culture eliminated the cytotoxicity of sterilized polystyrene and PU8. Hydrogen peroxide (H2O2) residues remaining in the sterilized materials were determined. Large amounts of H2O2 (5.1-186 micrograms) were detected in HEMA, PU8, Sil/PU6 and PMMA. In contrast, silicone and polyethylene contained low levels of H2O2. The amounts of residual H2O2 in the materials decreased with increasing aeration time, but the elimination rate of H2O2 differed among the test materials. The cytotoxic potential of the VPHP-sterilized materials correlated with the amounts of residual H2O2 present. These results indicated that the cytotoxicity of VPHP-sterilized materials was caused by the residual H2O2. To generalize the developed VPHP sterilization system for various medical devices, it is important to validate the aeration of materials for removal of cytotoxic residuals.

Evaluation of skin sensitization potential of nickel, chromium, titanium and zirconium salts using guinea-pigs and mice.

Contact sensitization capacity of four metal salts, nickel sulphate (NiSO4), potassium dichromate (K2Cr2O7), titanium chloride (TiCl4) and zirconium chloride (ZrCl4), was evaluated using guinea-pigs and mice. In the guinea-pig sensitization tests, we set up an injection concentration to 1% for all chemicals, and changed the challenge concentration. Guinea-pigs were sensitized with NiSO4, K2Cr2O7 and TiCl4. Among the test metal salts, K2Cr2O7 showed the highest sensitization rate and strongest skin reactions. ZrCl4 did not cause any sensitization responses under our experimental conditions. Minimum challenge concentration to cause a skin response was < 0.25% for K2Cr2O7, 0.5% for NiSO4 and 2% for TiCl4, respectively. A sensitive mouse lymph node assay (SLNA) also determined NiSO4 and K2Cr2O7 as a sensitizer. In the SLNA, TiCl4 caused mild lymph node responses, but was classified as a non-sensitizer as well as ZrCl4. Considering these results, the order of sensitization potential was K2Cr2O7 > NiSO4 > ZrCl4. NiSO4- and K2CrO7-sensitized animals did not show any reactions to ZrCl4 and TiCl4. No cross-reaction among these metal salts was found.

Correlations among chemical constituents, cytotoxicities and tissue responses: in the case of natural rubber latex materials.

Natural rubber latex films obtained from 40 different brands of rubber gloves were tested by quantitative chemical analyses, two cytotoxicity tests (agar-diffusion assay using L929 cells and colony assay using V79 cells) and 7-day implantation test in the rabbit muscle. Multiple regression analysis of these data showed that dithiocarbamate accelerators caused the toxicities whereas antioxidants did not. Thickness of inflammatory layer was the most useful parameter to evaluate tissue response among 13 histological parameters investigated. There were good correlations between the cytotoxicity indices and the thickness of inflammatory layer.